The p53 tumor suppressor, which acts as a transcription factor, controls a gene network that modulates cellular response to diverse stresses including DNA damage. Not surprisingly, p53 itself is exquisitely regulated through multiple signaling mechanisms that utilize protein-protein interactions and posttranscriptional modifications including ubiquitination, phosphorylation, and acetylation. p53 level and activity are normally restrained by its negative regulators Mdm2 (a p53 ubiquitin ligase) and Mdmx. Upon DNA damage, p53 is rapidly induced, via elevation of its stability, and activated, which often require the Ataxia-Telangiectasia Mutated kinase (ATM). Indeed, several recent studies have identified ATM-mediated phosphorylation of Mdm2 and Mdmx as an important mechanism that regulates p53 stabilization. However, it is not fully understood how p53 activation is controlled by ATM. In this proposal, we will investigate a novel ATM-mediated signaling mechanism - involving Ever Shorter Telomeres 1C (EST1C) - that regulates p53 stabilization and activation. Our preliminary studies show: (1) inactivation of EST1C abrogates p53 stabilization and activation and its function to block cell-cycle progression; (2) EST1C strongly promotes p53 acetylation; (3) DNA damage induces phosphorylation of EST1C and formation of a robust EST1C-p53 protein complex in an ATM- dependent fashion. Based on these results, we hypothesize that EST1C is a critical component in the ATM- p53 stress response pathway and plays an essential role in regulation of p53 function. To explore our hypothesis, we will use a multidisciplinary approach including tools in molecular biology, genetics, protein biochemistry, and knockout mouse models. In Aim 1, we will elucidate the signaling mechanism that regulates the interaction between EST1C and p53. In Aim 2, we will dissect the molecular mechanisms through which EST1C regulates p53. In Aim 3, we will determine the role of EST1C in regulation of p53 function in vivo. Completion of the proposed studies, which are outlined in the following three specific aims, will significantly advance our understanding of how the p53 network is regulated in stress response.